Identification of cholate as a shared substrate for the unidirectional efflux systems for methotrexate in L1210 mouse cells.
The bidirectional transport properties of cholate have been examined in leukemic L1210 mouse cells and compared with the transport of methotrexate. The cell entry of [3H]cholate was Na(+)-independent, linear with increasing concentrations of substrate, enhanced by decreasing pH, and uneffected by excess unlabeled cholate or by various anion-transport inhibitors and hence had the characteristics of passive diffusion or a pH-dependent mediated process with a high Kt for cholate. The efflux of [3H]cholate, however, could be attributed to carrier-mediated and energy-dependent transport. Efflux was rapid (t1/2 = 1.5 min) and could be increased with glucose and decreased with metabolic inhibitors, and it was inhibited by various compounds including bromosulfophthalein, probenecid, prostaglandin A1, reserpine, verapamil, quinidine, diamide, 1-methyl-3-isobutylxanthine and vincristine. The most potent inhibitor was prostaglandin A1, which reduced efflux by 50% at a concentration of 0.10 microM. Half-maximal inhibition by vincristine occurred at 4.8 microM. The maximum extent of inhibition with most of the inhibitors was 95%, although a lower value was observed with bromosulfophthalein (85%). When cholate efflux was compared with the efflux of methotrexate, both processes responded similarly to changes in the metabolic state of the cell. Moreover, the various inhibitors of cholate efflux also inhibited the efflux of methotrexate and the same concentration of each inhibitor was required for half-maximal inhibition of both processes. The efflux of folate and urate also proceeded via outwardly directed, unidirectional processes which were sensitive to bromosulfophthalein and probenecid. The results suggest that L1210 cells have the capacity for the unidirectional extrusion of cholate, methotrexate and probably other large, structurally dissimilar organic anions and that this efflux occurs via two or more very similar transport systems with a broad anion specificity. The function of an organic anion efflux system in vivo may be to facilitate the extrusion of cytotoxic metabolic anions which are too large to exit via the general anion-exchange carrier of these cells. Similarities in inhibitor specificity were also apparent between unidirectional anion efflux in L1210 cells and the drug efflux pump which is over-produced in cells with multidrug resistance.
Henderson
G B
GB
Department of Molecular and Experimental Medicine, Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Tsuji
J M
JM
D000818:Animals; D001692:Biological Transport; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002793:Cholic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D005492:Folic Acid / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D007939:Leukemia L1210 / Q000378:metabolism*; D008727:Methotrexate / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D011339:Probenecid / Q000494:pharmacology; D013379:Substrate Specificity; D013448:Sulfobromophthalein / Q000494:pharmacology; D013696:Temperature; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014527:Uric Acid / Q000378:metabolism; D014750:Vincristine / Q000494:pharmacology
D002793:Cholic Acids; D013448:Sulfobromophthalein; D014527:Uric Acid; D003571:Cytochalasin B; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D014750:Vincristine; D005492:Folic Acid; D005947:Glucose; D011339:Probenecid; D008727:Methotrexate